Occurrence of and Sequence Variation among F-Specific RNA Bacteriophage Subgroups in Feces and Wastewater of Urban and Animal Origins

Abstract

b F-specific RNA bacteriophages (FRNAPH) have been widely studied as tools for evaluating fecal or viral pollution in water. It has also been proposed that they can be used to differentiate human from animal fecal contamination. While FRNAPH subgroup I (FRNAPH-I) and FRNAPH-IV are often associated with animal pollution, FRNAPH-II and -III prevail in human wastewater. However, this distribution is not absolute, and variable survival rates in these subgroups lead to misinterpretation of the original distribution. In this context, we studied FRNAPH distribution in urban wastewater and animal feces/wastewater. To increase the specificity, we partially sequenced the genomes of phages of urban and animal origins. The persistence of the genomes and infectivity were also studied, over time in wastewater and during treatment, for each subgroup. FRNAPH-I genome sequences did not show any specific urban or animal clusters to allow development of molecular tools for differentiation. They were the most resistant and as such may be used as fecal or viral indicators. FRNAPH-II's low prevalence and low sequence variability in animal stools, combined with specific clusters formed by urban strains, allowed differentiation between urban and animal pollution by using a specific reverse transcription-PCR (RT-PCR) method. The subgroup's resistance over time was comparable to that of FRNAPH-I, but its surface properties allowed higher elimination rates during activated-sludge treatment. FRNAPH-III's low sequence variability in animal wastewater and specific cluster formation by urban strains also allowed differentiation by using a specific RT-PCR method. Nevertheless, its low resistance restricted it to being used only for recent urban pollution detection. FRNAPH-IV was too rare to be used. Water contamination by pathogens is a major public health problem. Microorganisms affecting water quality are mainly excreted in feces, and water contamination is caused by discharge of fecal waste of animal origin (e.g., from runoff after rain, farms, and slaughterhouses [SH]) or human origin (e.g., from raw or treated urban wastewater) into the environment. Enteric pathogens include bacteria, protozoa, and enteric viruses (1). The last have been identified in many cases of waterborne and food-borne diseases in developed countries (2).The virological quality of water is usually indirectly estimated from the number of fecal indicator bacteria, such as Escherichia coli and enterococci (3). However, these indicators have serious limitations, especially for the estimation of viral pollution. First, it has been largely demonstrated that viruses are more resistant than bacteria in the environment; therefore viral outbreaks may still be identified even in the absence of indicator bacteria (4, 5). Second, conventional indicators are present in human and animal feces, and they do not provide information about the origin of fecal contamination (6). Yet, this parameter is essential because the reservoir of many waterborne pathogens, especially viruses, is hu...

Waterborne Viruses and F-Specific Coliphages in Mixed-Use Watersheds: Microbial Associations, Host Specificities, and Affinities with Environmental/Land Use Factors

“…The stream systems studied had varied depth and substrates, which will contribute to there being a range in surface water temperatures affecting both persistence and viability. The lack of detection of F-RNA coliphage GIV in surface water found here is also in agreement with other studies (17,65,66).…”

“…In this study, F-RNA coliphage GI was the most frequent and most abundant genogroup detected, which was expected, given that the samples were predominantly from agriculturally and wildlife-impacted surface waters (12). Moreover, increased rates of survival for F-RNA coliphage GI compared to those of other F-RNA coliphage subgroups at different temperatures may contribute to a greater prevalence or persistence of F-RNA coliphage GI in surface water (17,30,33,66,67). The stream systems studied had varied depth and substrates, which will contribute to there being a range in surface water temperatures affecting both persistence and viability.…”

Relevance of F-Specific RNA Bacteriophages in Assessing Human Norovirus Risk in Shellfish and Environmental Waters

“…In this context, we compared the presence of FRNAPH and HuNoV genomes in water and shellfish samples, taking into account the aforementioned methodological limitations. Surrogates must behave as pathogenic viruses, and FRNAPH genome detection appears interesting due to their persistence being greater than infectivity (20)(21)(22)(23)65), allowing the source of fecal pollution to be tracked for a longer period of time, despite the presence of inactivating factors in the environment. Unfortunately, low genome levels in water require a concentration step, and poor yields and inhibition cause them to be around their detection limits.…”

“…RNA was then extracted using NucliSENS easyMAG (bioMérieux, Marcy l'Etoile, France) from 50 l of phage suspension, eluted in 100 l of buffer. Genotyping was then performed from RNA by a one-step multiplex quantitative RT-PCR, as used in a previous study (21).…”

“…Consequently, laboratories sometimes fail to detect the presence of HuNoV genomes in oysters involved in global HuNoV gastroenteritis outbreaks (17). On the other hand, a very high overestimation of the infectious risk may also occur (17)(18)(19) because of the persistence of viral genomes being greater than viral infectivity (20)(21)(22)(23), especially after disinfection treatment. It has been suggested that as shellfish are not able to bioaccumulate NoV RNA fragments present in seawater (24), the genomes detected in shellfish mainly result from infectious virus accumulation.…”